Electrophoresis, encompassing capillary electrophoresis (CE), micellar electrokinetic chromatography (MEKC) and microchip electrophoresis, is a very powerful technique for the analysis of analytes such as ions in a sample. Conventional capillary electrophoresis separates and detects charged ions of one polarity (i.e. anions or cations) through the application of a voltage potential to cause the charged ions to move through a separation capillary at different rates according to their electrophoretic mobility in the presence of a background electrolyte. Such conventional capillary electrophoresis is thus used to detect either the cations present in the sample, or the anions present in the sample. Where the analytes are neutral (uncharged) species, through control of the background electrolyte composition by, for example, the addition of a surfactant, these analytes can also be separated.
It has been recognised that it would be useful for anions and cations present in a sample to be analysed simultaneously. It would similarly be useful to be able to achieve, more generally, the separation and analysis of analytes through multiple separation channels or columns simultaneously.
The benefit of simultaneous analysis of anions and cations is clear; it negates the requirement for two separate analyses. In conventional CE, this is difficult because one of the charged species must migrate against the electroosmotic flow (EOF). It is possible to separate both but only when the EOF is greater than the electrophoretic mobility of the fastest target analyte of opposite polarity to the separation electrode. The practical drawback of this approach is that it is not suitable for the separation of the complete range of inorganic ions. With a cathodic EOF, this approach can separate the full range of cations, but is only suitable for low mobility anions. With an anodic EOF the reverse is true; it can separate the full range of anions but only low mobility cations. The peak capacity of the ions separated in a co-EOF manner is also compromised due to the speed at which they reach the detector.
There have been a small number of publications in recent times that seek to provide a technique for the simultaneous electrophoretic detection of cations and anions. One such technique relies on “dual-opposite end injection” (DOI-CE), in which the positively and negatively charged species are injected from opposite ends of a capillary. During electrophoretic analysis, which occurs under conditions of reduced EOF, analytes migrate from each end of the capillary, in opposite directions towards the detector located near the centre of the capillary. The drawback of this DOI-CE technique is that the separation space is reduced, so there must be precise control of the timing to ensure that anions and cations do not reach the detector at the same time.
Another method of simultaneous anion and cation analysis involves the use of an anionic complexing agent also being the anionic probe. Metal ions are converted to their chelated forms with EDTA or 2,6-pyridinedicarboxylate and separated from other anionic components under anionic separation conditions. Whilst this simplifies the system, this is only applicable to metals that can form an anionic complex and is not suitable for alkali and alkaline earths.
Other techniques considered previously suffer from other drawbacks, such as the requirement to load the sample at multiple points (i.e. multiple sample reservoirs), which then increases the size of the sample required for analysis, and complicates the design of the electrophoresis device and system. Additionally, such techniques rely on the application of a positive potential of differing magnitude at multiple locations in the apparatus, and grounding at two locations, which further complicates the design. Hydrodynamic suppression is achieved through hydrodynamic restrictors of a complicated design which restricts the ability for the apparatus to be created with commercially available equipment, and thus impacts on cost.
It is an object of the invention to provide an alternative technique for the simultaneous separation and detection of cations and anions in a sample. It is desired for the system to produce repeatable results, and to reflect the results that would be expected from two different analyses (for cations and anions) on the sample using conventional techniques. It is also desired for some embodiments to be based on a simple and robust design.
During the course of completing this analysis, it has also been found that the techniques allowing the simultaneous separation and detection of cations and anions can apply more generally to the separation and analysis of analytes (cations, anions or neutral species) through two separation columns via a single injection.
It has also been found that improved electrophoretic methods and systems could be achieved through developing new background electrolyte (or “buffer”) delivery options. Thus, according to some embodiments, it is an object to provide a new electrophoresis method and system for the separation of analytes in a sample which has a new degree of flexibility regarding the control of the background electrolyte. This has particular application to techniques that utilise two or more separation channels for the simultaneous separation of ions (e.g. cations in one channel, and anions in the other).